There is a need for techniques which can detect the presence of low concentrations of analytes with high accuracy, particularly when multiple analytes are present in a single sample. The need is particularly acute for detection of biomarkers and thus the diagnosis of the associated disease or disorder. One group of biomarkers which are difficult to detect in low concentrations are micro-ribonucleic acids (micro-RNA or miRNAs). miRNAs are highly stable RNA oligomers, which can regulate protein production post-transcriptionally. They act by one of two mechanisms. In plants, miRNAs have been shown to act chiefly by directing the cleavage of messenger RNA, whereas in animals, gene regulation by miRNAs typically involves hybridisdation of miRNAs to the 3′ UTRs of messenger RNAs, which hinders translation (Lee et al., Cell 75, 843-54 (1993); Wightman et al., Cell 75, 855-62 (1993); and Esquela-Kerscher et al., Cancer 6, 259-69 (2006)). miRNAs frequently bind to their targets with imperfect complementarity. They have been predicted to bind to as many as 200 gene targets each and to regulate more than a third of all human genes (Lewis et al., Cell 120, 15-20 (2005)).
The expression level of certain microRNAs is known to change in tumours, giving different tumour types characteristic patterns of microRNA expression (Rosenfeld, N. et al., Nature Biotechnology 26, 462-9 (2008)). In addition, miRNA profiles have been shown to be able to reveal the stage of tumour development with greater accuracy than messenger RNA profiles (Lu et al., Nature 435, 834-8 (2005) and Barshack et al., The International Journal of Biochemistry & Cell Biology 42, 1355-62 (2010)). These findings, together with the high stability of miRNAs, and the ability to detect circulating miRNAs in serum and plasma (Wang et al., Biochemical and Biophysical Research Communications 394, 184-8 (2010); Gilad et al., PloS One 3, e3148 (2008); and Keller et al., Nature Methods 8, 841-3 (2011)), have led to a considerable amount of interest in the potential use of microRNAs as cancer biomarkers. For treatment to be effective, cancers need to be classified accurately and treated differently, but the efficacy of tumour morphology evaluation as a means of classification is compromised by the fact that many different types of cancer share morphological features. miRNAs offer a potentially more reliable and less invasive solution.
The physiological concentration of microRNAs varies over a wide range, such as from low nM to fmol, and probably lower (Kirschner et al., PloS One 6, e24145 (2011) and the difference in expression level between different stages of cancers can be slight (Barshack et al., supra) so quantification with high sensitivity and accuracy is essential if a diagnostic test is to be useful.
Several methods have been used to identify and quantify miRNAs, such as Northern blotting (Reinhart et al., Genes & Development 16, 1616-26 (2002)), hybridisation to arrays (Chen et al., Nucleic Acids Research 36, e87 (2008) and Krichevsky et al., RNA 9, 1274-81 (2003)) and reverse transcription polymerase chain reaction, RT-PCR (Chen et al., Nucleic Acids Research 33, e179 (2005)). However, each approach has several shortcomings. For instance, Northern blots use a large quantity of total RNA, are not high throughput, are not readily multiplexed and are not strictly quantitative. Arrays have the advantage of being able to quantify many miRNAs at once but, as with Northern blots, lack sensitivity, need large amounts of RNA, or amplification, are not quantitative and also suffer from probe cross-hybridisation. Reverse transcriptase qPCR is considered to be inaccurate, is prone to primer cross-hybridisation if done in multiplex and results are often inconsistent between different laboratories (Murphy et al., Expert Review of Molecular Diagnostics 9, 187-97 (2009)). Furthermore, all of these methods require the use of RNA extraction kits to isolate and concentrate total RNA, which is inconvenient.
The ideal method for miRNA quantification would by highly sensitive, would not require a large amount of starting material, would be high throughput, capable of multiplexed measurement, would not require amplification of the RNA and could be performed directly on blood, serum or plasma. A small number of methods have been described which are capable of detecting and quantifying miRNA directly from cell lysate including surface Plasmon resonance and protein facilitated affinity capillary electrophoresis (Cissell et al., Analytical Chemistry 80, 2319-25 (2008); Nasheri et al., Analytical Biochemistry 412, 165-72 (2011); and Khan et al., Analytical Chemistry 83, 6196-201 (2011)), but these approaches require expensive hardware and have not been demonstrated to have multiplexing capability.
Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for a variety of analytes, such as polymers and small molecules. When a potential is applied across a nanopore, there is a change in the current flow when a molecule, such as a nucleotide or a polynucleotide, resides transiently in the barrel or channel of the nanopore for a certain period of time. Specific molecules, such as specific nucleotides and specific polynucleotides, give current changes of known signature and duration. Such current changes can be used to identify the nucleotide or polynucleotide present in the pore.